CN101886088B - Transgenic structure and transgenic plant - Google Patents

Abstract

The invention relates to a transgenic structure and a transgenic plant. The transgenic structure is used for expressing glyphosate-tolerant 5-enoplyrul-shikimate-3-phosphate synthase in plants. The transgenic plant shows glyphosate tolerance.

Description

Transgenic constructs and transgenic plant

Technical field

Usually, the present invention relates to molecular biology and transgenic plant field.Particularly, the present invention relates to for the transgenic constructs of expressing careless glycosides phosphine (glyphosate) tolerance type 5-enol pyruvylshikimate-3-phosphate synthase (5-enolpyrul-shikimate-3-phosphate synthase, EPSPS) at vegetable cell, comprise the recombinant vectors of described transgenic constructs and with host cell, transgenic plant cells and transgenic plant of described transgenic constructs or recombinant vectors conversion and preparation method thereof.The invention still further relates to and give plant with the chemical sproof method of careless glycosides phosphine.

Background technology

EPSPS is the key enzyme in the die aromatischen Aminosaeuren route of synthesis, is present in plant and the bacterium.Grass glycosides phosphine, namely the N-phosphonomethylglycine is weedicide after a kind of wide spectrum, the efficiently germination.It is the competitive inhibitor of the synthetic substrate phosphoenolpyruvic acid (PEP) of EPSPS, these two kinds of substrates of PEP capable of blocking and 3-phosphoric acid shikimic acid under EPSPS catalysis to the conversion of 5-enol pyruvylshikimate-3-phosphoric acid shikimic acid, thereby the route of synthesis of the synthetic precursor-shikimic acid of blocking-up die aromatischen Aminosaeuren causes plant and bacterium dead.

Plant can be by obtaining to the careless glycosides phosphine tolerance type EPSPS encoding gene of the stable introducing of their genome to the resistance of careless glycosides phosphine.Known careless glycosides phosphine tolerance type EPSPS gene mainly contains two large classes at present: and the I class (for example US 4,971, and 908; US 5,310, and 667; US 5,866,775 etc.) and II class (US5 for example, 627,061; US 5,633, and 435 etc.), they all successfully import in the Plant Genome, have obtained to have the vegetable cell of careless glycosides phosphine tolerance, and complete plant.

The objective of the invention is stable or the expression of raising EPSPS transgenosis in vegetable cell, thereby obtain to tolerate the transgenic plant of careless glycosides phosphine.

Summary of the invention

The invention provides a kind of polynucleotide of separation, its direction with from 5 ' to 3 ' comprises the nucleotide sequence of coding chloroplast transit peptides (chloroplast transfer peptide, CTP) and the nucleotide sequence of the careless glycosides phosphine tolerance type EPSPS of coding.In one embodiment, the nucleotide sequence of described coding CTP is the nucleotide sequence of coding Arabidopis thaliana EPSPS CTP, for example sequence shown in the sequence 7 in the sequence table.In another embodiment, the nucleotide sequence of the careless glycosides phosphine of described coding tolerance type EPSPS is that coding pseudomonas putida (Pseudomonas putida) EPSPS is the nucleotide sequence of AroA, for example sequence shown in the sequence 9 or 10.

Perhaps, can modify the nucleotide sequence of coding CTP and/or the nucleotide sequence of the careless glycosides phosphine tolerance type EPSPS that encodes, for example the disappearance of one or several codon (being nucleotide triplet) and/or interpolation and/or alternative.In one embodiment, described modification is reticent, for example the nucleotide sequence encoding amino acid sequence EPSPS identical with careless glycosides phosphine tolerance type EPSPS natural acid sequence of the coding grass glycosides phosphine tolerance type EPSPS of process modification.In a specific embodiment, described modification be for the vegetable codon preferences and/or reduce the nucleic acid secondary structure and carry out codon optimized.In another embodiment, described modification is guarded, for example pass through the nucleotide sequence encoding amino acid sequence EPSPS variant different from careless glycosides phosphine tolerance type EPSPS natural acid sequence of the coding grass glycosides phosphine tolerance type EPSPS that modifies, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.Modification non-silence or nonconservative is also contained in the present invention.

Or, can modify the nucleotide sequence of coding CTP and/or the nucleotide sequence of the careless glycosides phosphine tolerance type EPSPS that encodes, for example the disappearance of one or several codon (being nucleotide triplet) and/or interpolation and/or alternative.In one embodiment, nucleotide sequence to the careless glycosides phosphine tolerance type EPSPS that encodes is modified, and has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% nucleotide sequence homology or identity through the natural nucleus glycoside acid sequence of the nucleotide sequence modified and the careless glycosides phosphine tolerance type EPSPS of coding.A specific embodiment, the described nucleotide sequence encoding amino acid sequence EPSPS identical with careless glycosides phosphine tolerance type EPSPS natural acid sequence through modifying.In another specific embodiment, the described nucleotide sequence encoding amino acid sequence EPSPS variant different from careless glycosides phosphine tolerance type EPSPS natural acid sequence through modifying, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.In another embodiment, nucleotide sequence to the careless glycosides phosphine tolerance type EPSPS that encodes is modified, through the nucleotide sequence encoding amino acid sequence EPSPS variant different from careless glycosides phosphine tolerance type EPSPS natural acid sequence of modifying, the natural acid sequence of the aminoacid sequence of described EPSPS variant and careless glycosides phosphine tolerance type EPSPS has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence homology or identity, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.

Or, can modify the nucleotide sequence of coding CTP and/or the nucleotide sequence of the careless glycosides phosphine tolerance type EPSPS that encodes, for example the disappearance of one or several codon (being nucleotide triplet) and/or interpolation and/or alternative.In one embodiment, nucleotide sequence to the careless glycosides phosphine tolerance type EPSPS that encodes is modified, and the fragment of the natural nucleus glycoside acid sequence of the nucleotide sequence that process is modified and the careless glycosides phosphine tolerance type EPSPS of coding is (for example at least about 10, about 15, about 20, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, the fragment of about 500 or more continuous nucleotides) (for example very low at stringent condition, low, medium, high, or very high stringent condition) lower hybridization.A specific embodiment, the described nucleotide sequence encoding amino acid sequence EPSPS identical with careless glycosides phosphine tolerance type EPSPS natural acid sequence through modifying.In another specific embodiment, the described nucleotide sequence encoding amino acid sequence EPSPS variant different from careless glycosides phosphine tolerance type EPSPS natural acid sequence through modifying, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.In another embodiment, nucleotide sequence to the careless glycosides phosphine tolerance type EPSPS that encodes is modified, through the nucleotide sequence encoding amino acid sequence EPSPS variant different from careless glycosides phosphine tolerance type EPSPS natural acid sequence of modifying, the natural acid sequence of the aminoacid sequence of described EPSPS variant and careless glycosides phosphine tolerance type EPSPS has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence homology or identity, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.

The invention provides a kind of fusion rotein, it comprises CTP and careless glycosides phosphine tolerance type EPSPS with the direction of holding the C end from N.In one embodiment, described CTP is Arabidopis thaliana EPSPS CTP, for example sequence shown in the sequence 6 in the sequence table.In another embodiment, described careless glycosides phosphine tolerance type EPSPS is that pseudomonas putida EPSPS is the aminoacid sequence of AroA, for example sequence shown in the sequence 8.

Perhaps, can modify the aminoacid sequence of CTP and/or the aminoacid sequence of careless glycosides phosphine tolerance type EPSPS, for example the disappearance of one or several amino-acid residue and/or interpolation and/or alternative.In one embodiment, described modification is guarded, for example the aminoacid sequence of the careless glycosides phosphine tolerance type EPSPS of process modification is different from careless glycosides phosphine tolerance type EPSPS natural acid sequence, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.In a specific embodiment, the natural acid sequence of the aminoacid sequence of described EPSPS variant and careless glycosides phosphine tolerance type EPSPS has about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence homology or identity, and described EPSPS variant has the active and careless glycosides phosphine tolerance of the EPSPS identical or suitable with natural EPSPS.Nonconservative modification is also contained in the present invention.

The present invention also provides a kind of nucleic acid construct, and it comprises the polynucleotide of separation of the present invention.In one embodiment, described nucleic acid construct is expression cassette.The present invention also provides a kind of recombinant vectors, and it comprises the polynucleotide of separation of the present invention, or comprises nucleic acid construct of the present invention.In one embodiment, described recombinant vectors is recombinant clone plasmid or recombinant expression plasmid.The present invention also provides a kind of host cell, and it comprises the polynucleotide of separation of the present invention, or comprises nucleic acid construct of the present invention, or comprises recombinant vectors of the present invention.In one embodiment, described host cell is prokaryotic host cell, for example bacterial host cell.In a specific embodiment, described bacterial host cell is intestinal bacteria (Escherichia coli) host cells.In another specific embodiment, described bacterial host cell is the Agrobacterium host cell, such as agrobacterium tumefaciens (Agrobacterium tumefaciens) host cell.

The present invention also provides a kind of transgenic plant cells and transgenic plant, and it comprises the polynucleotide of separation of the present invention, or comprises nucleic acid construct of the present invention, or comprises recombinant vectors of the present invention, or comprises host cell of the present invention.In one embodiment, the polynucleotide of separation of the present invention are incorporated in the genome of described transgenic plant cells.In one embodiment, described plant or described vegetable cell are farm crop, comprise food crop and cash crop.In one embodiment, described plant or described vegetable cell are for example to be selected from lower group plant or vegetable cell: rice (Oryza, comprise OryzasativaL.), millet (Setaria italicaL.), wheat (Triticum, comprise Triticum sestivumL.), barley (Hordeum, comprise Hordeum vulgare L.), oat (Avena, comprise Avena sativaL.), jowar (Sorghum, comprise Sorghum bicolor L.), corn (Zea, comprise Zea may L.), potato (Solanum tuberosum L.), soybean (Glycine, comprise Glycine max L.), peanut (Arachis, comprise Arachis hypogaea L.), Sunflower Receptacle (Helianthus, comprise Helianthusannuus L.), cotton (Gossypium, comprise Gossypium hirsutum L.), clover (Medicago, comprise Medicago sativa L.), tobacco (Nicotiana comprises Nicotiana tabacum L.), rape (Brassica campestris L.), green vegetables (Brassica chinensis L.), wild cabbage (Brassicaoleracea L.), Cauliflower (Brassica oleracea var.botrytis L.), Caulis et Folium Brassicae capitatae (Brassicaoleracea var.capitata L.) etc.In one embodiment, described transgenic plant cells and transgenic plant have the tolerance to careless glycosides phosphine.

The present invention also provides the method for preparing transgenic plant cells of the present invention and transgenic plant, and it comprises the step that the polynucleotide of separation of the present invention, nucleic acid construct, recombinant vectors or recombinant host cell is imported vegetable cell.In one embodiment, the polynucleotide of separation of the present invention are incorporated in the genome of described transgenic plant cells.In one embodiment, described transgenic plant cells and transgenic plant have the tolerance to careless glycosides phosphine.

The present invention also provides and has given vegetable cell or the plant method with careless glycosides phosphine tolerance, and it comprises the step that the polynucleotide of separation of the present invention, nucleic acid construct, recombinant vectors or recombinant host cell is imported vegetable cell.In one embodiment, the polynucleotide of separation of the present invention are incorporated in the genome of described transgenic plant cells.

Description of drawings

Fig. 1 is the synoptic diagram of transgene expression cassette.LB, the left margin of T-DNA; RB, the right margin of T-DNA; Pnos, the promotor of rouge alkali synthetase gene Nos; P35S, the 35S promoter of tobacco mosaic virus (TMV), i.e. CaMV35S promotor; Tnos, the promotor of rouge alkali synthetase gene Nos; NptII, neomycin phosphotransferase II gene; CTP derives from the encoding sequence of chloroplast(id) leading peptide of the EPSP synthase of Arabidopis thaliana; N-EPSPS derives from the encoding sequence of the EPSP synthase of pseudomonas putida; O-EPSPS, the encoding sequence of the EPSP synthase after codon optimized (artificial synthesized sequence that designs according to the codon-bias of dicotyledons).

Fig. 2 has shown the upgrowth situation after transgene tobacco and wild-type tobacco spray careless glycosides phosphine weedicide.A: transgene tobacco, B: wild-type tobacco.

Fig. 3 has shown that the PCR of transgene tobacco detects.Swimming lane 1-6: be followed successively by 2323-4/5/15/16/20/23; Swimming lane M:DNA mark DL2000; Swimming lane WT: wild-type contrast tobacco.

Fig. 4 has shown that the Southern trace of transgene tobacco detects.Swimming lane 1-4: be followed successively by 2323-4/5/15/16; Swimming lane 5: wild-type contrast tobacco.

Fig. 5 has shown that the RT-PCR of transgene tobacco detects.Swimming lane M:DNA mark DL2000; Swimming lane WT: wild-type contrast tobacco; Swimming lane 1-6: be followed successively by 2323-4/5/15/16/20/23.

Fig. 6 has shown that the RT-PCR of the codon optimized front and back of transgene tobacco detects.Swimming lane 1: codon optimized transgene tobacco 2323-4; Swimming lane 2: the Actin muscle confidential reference items among the codon optimized transgene tobacco 2323-4; Swimming lane 3: the Actin muscle confidential reference items among the transgene tobacco 2349-2 that codon is not optimized; Swimming lane 4: the transgene tobacco 2349-2 that codon is not optimized.

Fig. 7 has shown that the Western trace of the EPSPS albumen of transgene tobacco detects.Swimming lane 1: the EPSPS of pseudomonas putida; Swimming lane 2-7: be followed successively by 2323-4/5/15/16/20/23; Swimming lane 8: non-transgenic tobacco contrast; Swimming lane M: protein markers.

Fig. 8 has shown the careless glycosides phosphine tolerance mensuration of wild-type tobacco.The rudiment situation of wild-type tobacco seed on the flat board that contains different concns grass glycosides phosphine: from left to right, the concentration of careless glycosides phosphine is followed successively by 0mM, 0.05mM, 0.1mM, 0.2mM and 0.5mM.

Fig. 9 has shown that transgene tobacco T0 measures for seed germination.A: do not contain the seed germination situation on the MS flat board of careless glycosides phosphine; B: contain the seed germination situation on the MS flat board of 1mM grass glycosides phosphine.Left one side of something of every flat board is empty carrier contrast tobacco, and right one side of something is the transgene tobacco of the careless glycosides phosphine of tolerance.

Figure 10 has shown that transgene tobacco T0 measures for seed germination: when a. grass glycosides phosphine concentration was 0mM, when the equal energy of the seedling of transgene tobacco sprout growth b. grass glycosides phosphine concentration was 10mM, the part seedling of transgene tobacco tolerated careless glycosides phosphine.

Figure 11 has shown that the PCR in transgene tobacco T1 generation detects.Swimming lane 1-10: the T1 of transgene tobacco is for plant; Swimming lane M:DNA mark DL2000; Swimming lane 12: non-transgenic tobacco contrast; Swimming lane 13: plasmid positive control.

Detailed Description Of The Invention

Definition

" sequence homology or identity % " refers to contrast by sequence herein, and insert in case of necessity the space with the largest percentage that obtains homology or identity (and, when sequence of calculation identity %, do not consider any conservative substituting), be same as the percentage ratio of the amino-acid residue of target sequence in the candidate sequence.Sequence described herein comprises aminoacid sequence and nucleotide sequence.Can use the whole bag of tricks known in the art to measure sequence homology or identity per-cent, for example, use the available computer software of the public such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR).Those skilled in the art can be identified for the design parameter of sequence contrast, and the more required any algorithm of the high specific of full length sequence.

" severity " of hybridization can be easy to determine by those of ordinary skills, and usually calculate by rule of thumb according to probe length, wash temperature and salt concn.Generally speaking, the temperature that the probe of growing is had relatively high expectations is with correct annealing, and short probe needs lower temperature.Hybridization usually depends on when complementary strand and is present in the ability that time variation DNA anneals again in the environment that is lower than its melting temperature(Tm).But the expectation identity degree between probe and the hybridization sequences is higher, and spendable relative temperature is also higher.The result is infer that higher relative temperature will trend towards making reaction conditions more strict, and lesser temps to be just not strict yet.About other details and the explanation of hybridization severity, referring to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995).

" high stringency " is by following every definition: (1) adopts low ionic strength and high temperature to clean; 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate is in 50 ℃; (2) in crossover process, adopt denaturing agent; 50% (v/v) methane amide and 0.1% bovine serum albumin(BSA)/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer pH 6.5 and 750mM sodium-chlor, the 75mM Trisodium Citrate is in 42 ℃; Or (3) adopt 50% methane amide, 5x SSC (0.75MNaCl, 0.075M Trisodium Citrate), 50mM sodium phosphate (pH 6.8), 0.1% trisodium phosphate, 5xDenhardtShi solution, the salmon sperm dna of supersound process (50 μ g/ml), 0.1%SDS, with 10% sulfuric acid dextran, in 42 ℃, and clean in 0.2x SSC (sodium chloride/sodium citrate) and 50% methane amide in 42 ℃, then in 55 ℃ of 0.1x SSC that containing EDTA, carry out high severity cleaning.

" medium stringent condition " can be such as Sambrook et al., Molecular Cloning:ALaboratory Manual, New York, identify described in the Cold Spring Harbor Press (1989), be included in 37 ℃ and containing 20% methane amide, 5x SSC (150mM NaCl, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x DenhardtShi solution, 10% sulfuric acid dextran, and be incubated overnight in the solution of the salmon sperm dna of 20mg/ml sex change shearing, then in about 37-50 ℃ of cleaning filter membranes in 1x SSC.The technician will recognize and adjust how where necessary temperature, ionic strength etc. to adapt to such as factors such as probe length.

" nucleic acid construct " and " transgenic constructs " synonym refer to strand or double chain acid molecule herein, and these molecular separation are from natural gene or contained combination and nucleic acid fragment arranged side by side in the non-existent mode of nature through change.When nucleic acid construct contains when expressing required all regulating and controlling sequences of EPSPS of the present invention, term " nucleic acid construct " is synonym with " expression cassette ".

" regulating and controlling sequence " comprises all necessary or favourable elements of expression polypeptide of the present invention herein.Each regulating and controlling sequence both can be that the nucleotide sequence of this polypeptide of coding naturally has, and also can be external.This regulating and controlling sequence includes, but not limited to leader sequence, polyadenylation sequence, propeptide sequence, promotor and transcription terminator.Regulating and controlling sequence is minimum should to be comprised promotor and transcribes and the translation termination signal.In order to import special restriction enzyme site to help regulating and controlling sequence to connect with the coding region of the nucleotide sequence of the heterologous polypeptide of being connected, can prepare the regulating and controlling sequence with joint.

" being operably connected " herein is defined as the nucleotide sequence that makes separation of the present invention and links to each other with any other sequence with respect to this sequence homology or allos, thus make they do as a whole can a kind of product of coding.When needing, can pass through certain way, as cutting by the enzyme of restriction enzyme they be separated.Homology described here or heterologous sequence can be any sequences, if any regulating and controlling sequence that instructs the nucleotide sequence of separation of the present invention in particular host cell, to express, or with the sequence of a kind of fusion rotein of the common coding of the nucleotide sequence that separates of the present invention, etc.

" host cell " comprises any nucleotide sequence that can accept separation of the present invention herein, maybe can accept to contain construct or the carrier of this sequence, and make this sequence stable maintenance at intracellular cell.After host cell has comprised the nucleotide sequence of separation of the present invention, can obtain proterties or feature by this sequence encoding.

EPSPS

The protein that one aspect of the present invention relates to the careless glycosides phosphine of tolerance and has the EPSPS activity, for example EPSPS of pseudomonas putida, i.e. AroA, for example sequence in the sequence table 8.

The invention still further relates to variant or the mutant of above-mentioned protein, the aminoacid sequence of wherein said variant or mutant has one or several amino acid whose replacement, disappearance and/or interpolation with respect to aminoacid sequence described herein, but this variant or mutant still tolerate careless glycosides phosphine and have the EPSPS activity.Described amino acid whose replacement, disappearance and/or interpolation all are that this area routine techniques can be finished.Preferred this seed amino acid is changed to: little characteristic changing, i.e. and the folding and/or active conserved amino acid of not remarkably influenced albumen replaces; Little disappearance, usually about 1-30 amino acid whose disappearance; Little amino or carboxyl terminal extend, and for example aminoterminal extends a methionine residues; Little connection peptides, for example about 20-25 residue is long.

The conservative example that replaces is the replacement that occurs in following amino acid group: basic aminoacids is (such as arginine, Methionin and Histidine), acidic amino acid (such as L-glutamic acid and aspartic acid), polare Aminosaeren is (such as glutamine, l-asparagine), hydrophobic amino acid is (such as leucine, Isoleucine and α-amino-isovaleric acid), aromatic amino acid is (such as phenylalanine, tryptophane and tyrosine), and small molecules amino acid is (such as glycine, L-Ala, Serine, Threonine and methionine(Met)).Usually those aminoacid replacement that do not change given activity are well-known in this area, and by, for example, N.Neurath and R.L.Hill are described in the Protein of new york academic press (Academic Press) in 1979 publication.Modal exchange has Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, and their opposite exchanges.

For a person skilled in the art clearly, this replacement can occur outside the zone that molecular function is played an important role, and still produces active polypeptide.For the polypeptide by the nucleic acid sequence encoding of separation of the present invention, it is active essential and therefore select not substituted amino-acid residue, can be according to methods known in the art, as site-directed mutagenesis or alanine scanning mutagenesis identify (as referring to, Cunningham and Wells, 1989, Science 244:1081-1085).A rear technology is that each positively charged residue place introduces sudden change in molecule, and the careless glycosides phosphine tolerance type EPSPS that detects the gained mutating molecule is active, thus definite amino-acid residue that this molecular activity is overstated and wanted.Substrate-enzyme interacting site also can be measured by the analysis of its three-dimensional structure, this three-dimensional structure can by the technical measurements such as nuclear magnetic resonance spectroscopy, crystallography or light affinity tag (referring to, such as de Vos etc., 1992, Science 255:306-312; Smith etc., 1992, J.Mol.Biol 224:899-904; Wlodaver etc., 1992, FEBS Letters 309:59-64).

The invention still further relates to the aminoacid sequence that has certain homology or identity with aminoacid sequence described herein (for example aminoacid sequence shown in the sequence 8), be at least about 50% such as homology or identity, preferably at least about 60%, preferably at least about 65%, preferably at least about 66%, preferably at least about 67%, preferably at least about 68%, preferably at least about 69%, preferably at least about 70%, preferably at least about 71%, preferably at least about 72%, preferably at least about 73%, preferably at least about 74%, preferably at least about 75%, preferably at least about 76%, preferably at least about 77%, preferably at least about 78%, preferably at least about 79%, more preferably at least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, most preferably at least about 95%, most preferably at least about 96%, most preferably at least about 97%, most preferably at least about 98%, at least about 99%, be careless glycosides phosphine tolerance type EPSPS albumen as long as have the albumen of described aminoacid sequence most preferably, this sequence just belongs to scope of the present invention.

The invention still further relates to following protein, its have the EPSPS that contains aminoacid sequence described herein (for example aminoacid sequence shown in the sequence 8) activity at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 100%.

Another aspect of the present invention relates to the coding careless glycosides phosphine of tolerance and has the polynucleotide of the protein (for example EPSPS of pseudomonas putida, i.e. AroA) of EPSPS activity, for example sequence in the sequence table 9 or 10.

The invention still further relates to variant or the mutant of above-mentioned polynucleotide, the nucleotide sequence of wherein said variant or mutant is shown replacement, disappearance and/or the interpolation of one or several triplet codon with respect to nucleotides sequence described herein, but the protein that this variant or mutant are still encoded the careless glycosides phosphine of tolerance and had the EPSPS activity.The replacement of described triplet codon, disappearance and/or interpolation all are that this area routine techniques can be finished.Preferred this triplet codon variation causes following amino acid to change: little characteristic changing, i.e. and the folding and/or active conserved amino acid of not remarkably influenced albumen replaces; Little disappearance, usually about 1-30 amino acid whose disappearance; Little amino or carboxyl terminal extend, and for example aminoterminal extends a methionine residues; Little connection peptides, for example about 20-25 residue is long.

The invention still further relates to the nucleotide sequence that has certain homology or identity with nucleotide sequence described herein (for example nucleotide sequence shown in the sequence 9 or 10), be at least about 65% such as homology or identity, preferably at least about 66%, preferably at least about 67%, preferably at least about 68%, preferably at least about 69%, preferably at least about 70%, preferably at least about 71%, preferably at least about 72%, preferably at least about 73%, preferably at least about 74%, preferably at least about 75%, preferably at least about 76%, preferably at least about 77%, preferably at least about 78%, preferably at least about 79%, more preferably at least about 80%, more preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, most preferably at least about 95%, most preferably at least about 96%, most preferably at least about 97%, most preferably at least about 98%, most preferably at least about 99%, as long as have the polynucleotide encoding grass glycosides phosphine tolerance type EPSPS albumen of described nucleotide sequence, this sequence just belongs to scope of the present invention.

The invention still further relates to following polynucleotide, the protein of its coding have the EPSPS that contains aminoacid sequence described herein (for example aminoacid sequence shown in the sequence 8) activity at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 100%.

The polynucleotide of separation of the present invention can obtain from the nature clone according to method well-known in the art.Cloning process can comprise: restriction enzyme cutting and separation contain the expection nucleic acid fragment of the nucleotide sequence of the target protein of encoding, in this fragment insertion vector molecule, this recombinant vectors is incorporated in the host cell, copies thereby make a plurality of copies of this nucleotide sequence or be cloned in this host cell.But, easier method is, according to nucleotide sequence disclosed herein, sequence as described in synthetic with automatic Nucleotide synthesizer (such as the ABI394 dna synthesizer of AppliedBiosystems company), or with reference to October in 2000 disclosed Chinese patent application 99103472.4 on the 4th method, then the fragment of synthetic above-mentioned nucleotide sequence connects into complete sequence with conventional ligase enzyme and carrier with these fragments respectively.

Nucleotide sequence of the present invention can be sequence or its any composition of genome sequence, cDNA sequence, RNA sequence, semisynthetic sequence, complete synthetic.

When suitable, nucleotide sequence that can Optimized Coding Based EPSPS is to improve the expression in plant.In other words, can come with the codon of plant-preference the nucleotide sequence of composite coding EPSPS, express to improve.Referring to for example Campbell and Gowri (1990) Plant Physiol.92:1-11.The method of synthetic plant-preference type gene is well known in the art.Referring to for example U.S. Patent number 5,380,831; 5,436,391; And Murray etc., (1989) Nucleic Acids Res.17:477-498.

Can also be for the secondary structure that reduces nucleic acid the nucleotide sequence of Optimized Coding Based EPSPS, thereby the expression that improves gene.

Nucleic acid construct or transgenic constructs

Another aspect of the present invention relates to nucleic acid construct.

In one embodiment, described nucleic acid construct is used for expressing careless glycosides phosphine tolerance type EPSPS and expressed careless glycosides phosphine tolerance type EPSPS being positioned chloroplast(id) at vegetable cell.When target gene is not when expressing in chloroplast(id), nucleic acid construct will additionally comprise the nucleotide sequence of the chloroplast transit peptides of encoding, and arrive chloroplast(id) to instruct the target gene product.This type of chloroplast transit peptides is known in the art.Referring to such as von Heijne etc., (1991) Plant Mol.Biol.Rep.9:104-126; Clark etc., (1989) J.Biol.Chem.264:17544-17550; Della-Cioppa etc., (1987) Plant Physiol.84:965-968; Romer etc., (1993) Biochem.Biophys.Res.Commun.196:1414-1421; And Shah etc., (1986) Science 233:478-481.Other transit peptides comprises the transhipment skin described in U. S. application numbers 20020073443 and the U. S. application numbers 20020178467.In other embodiments, target gene can be navigated to component place in extracellular or other the cell, for example nuclear, plastosome or endoplasmic reticulum by target.

The sequence of target chloroplast(id) is known in the art, comprises ribulose-1,5-bisphosphate, the chloroplast(id) small subunit of 5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho etc., (1996) Plant Mol.Biol.30:769-780; Schnell etc., (1991) J.Biol.Chem.266 (5): 3335-3342); EPSPS (Archer etc., (1990) J.Bioenerg.Biomemb.22 (6): 789-810); Tryptophan synthetase (Zhao etc., (1995) J.Biol.Chem.270 (11): 6081-6087); Plastocyanin (Lawrence etc., (1997) J.Biol.Chem.272 (33): 20357-20363); With light harvesting chlorophyll a/b in conjunction with albumen (LHBP) (Lamppa etc., (1988) J.Biol.Chem.263:14996-14999).Also referring to von Heijne etc., (1991) PlantMol.Biol.Rep.9:104-126; Clark etc., (1989) J.Biol.Chem.264:17544-17550; Della-Cioppa etc., (1987) Plant Physiol.84:965-968; Romer etc., (1993) Biochem.Biophys.Res.Commun.196:1414-1421; And Shah etc., (1986) Science233:478-481.

The method that transforms chloroplast(id) is known in the art.Referring to such as Svab etc., (1990) Proc.Natl.Acad.Sci.USA 87:8526-8530; Svab and Maliga, (1993) Proc.Natl.Acad.Sci.USA 90:913-917; Svab and Maliga, (1993) EMBO J.12:601-606.

In one embodiment, nucleic acid construct of the present invention comprises the nucleotide sequence of the nucleotide sequence of the CTP that encodes and the careless glycosides phosphine tolerance type EPSPS that encodes with from 5 ' to 3 ' direction.For example, the nucleotide sequence of described coding CTP can be the nucleotide sequence of coding Arabidopis thaliana EPSPS CTP, for example sequence in the sequence table 6.For example, the nucleotide sequence of the careless glycosides phosphine of described coding tolerance type EPSPS can be the nucleotide sequence of coding pseudomonas putida EPSPS gene aroA, for example sequence in the sequence table 9 or 10.

Nucleic acid construct of the present invention also can contain can make above-mentioned sequence express necessary regulating and controlling sequence in selected host cell.Described regulating and controlling sequence is operably connected in nucleic acid construct with the above-mentioned nucleotide sequence that separates.

Regulating and controlling sequence can be promoter sequence, comprises the transcription regulating nucleotide sequence of the expression that mediates polypeptide.Promotor can be any nucleotide sequence that shows transcriptional activity in cell, comprise sudden change, brachymemma, as to reach heterozygosis promotor, and can be outside Codocyte or in the cell obtain the gene of polypeptide, these polypeptide can with this cell homology or non-homology.Numerous promotors for prokaryotic cell prokaryocyte are known in the art.

Regulating and controlling sequence also can be suitable transcription termination sequence, is namely identified the sequence that stops transcribing by host cell as herein described.This terminator sequence is operably connected with 3 ' end of the nucleotide sequence of coded polypeptide.Any have the terminator of function to use in the present invention in host cell.

Regulating and controlling sequence can be suitable leader sequence, i.e. the upper non-translational region very important to the translation of cell of mRNA.Leader sequence is operably connected with 5 ' end of the nucleotide sequence of coded polypeptide.Any have the leader sequence of function to use in the present invention in host cell.

Regulating and controlling sequence also can be the polyadenylation sequence.This sequence is operably connected with 3 ' end of nucleotide sequence, and when transcribing, is identified as signal by cell, thereby the polyadenylic acid residue is added on the mRNA that transcribes out.Any have the polyadenylation sequence of function to use in the present invention in this cell.

Nucleic acid construct can also comprise the nucleotide sequence that one or several is such, one or several is conducive to the factor that directing heterologous polypeptide is expressed these nucleic acid sequence encodings, for example, activating transcription factor (such as trans-acting factor), chaperone and processed-type proteolytic enzyme.Any host cell particularly in bacterial cell and the vegetable cell effectively the factor can use in the present invention.The nucleic acid of one or several these factors of encoding is not necessarily connected with the nucleotide sequence of coding heterologous polypeptide.

Recombinant vectors or recombinant expression vector

Above-mentioned various nucleic acid and regulating and controlling sequence can be connected on the conventional carriers such as plasmid or virus; produce " recombinant expression vector " of the present invention; method therefor be well-known to those skilled in the art (can be referring to J.Sambrook; E.F.Fritsch and T. Maniatus, 1989, Molecullar Cloning; laboratorymannual; the 2nd edition, Cold Spring, NY).Described carrier can comprise one or several easily restriction endonuclease sites.The compatibility of carrier and used host cell is depended in the selection of carrier usually.Described carrier can be linearity or closed hoop plasmid.Such carrier can be self-replicating type carrier, that is, with the carrier that the form of the outer entity of karyomit(e) exists, it copies and is independent of chromosomal copying, such as plasmid (a kind of extra-chromosomal element), minichromosome, or artificial chromosome.Carrier can comprise any means of guaranteeing self-replacation.Perhaps, described carrier also can behind transfered cell, be incorporated in the genome and with karyomit(e) and copy.Carrier system can be single carrier or plasmid or two or more carriers or plasmid (they comprise target nucleic acid sequence jointly), or transposon.

In the situation that carrier is integrated to cellular genome, this carrier can comprise the additional nucleotide sequence that instructs this carrier to be integrated into cellular genome by homologous recombination.These additional nucleotide sequences can make this carrier be integrated in the genome in chromosomal exact position.The possibility of integrating in order to be increased in the exact position, integrated element should preferably comprise the nucleic acid of sufficient amount, 100 to 1500 base pairs for example, preferred 400 to 1500 base pairs, 800 to 1500 base pairs most preferably, their its corresponding target sequence height homologies are to improve the possibility of homologous recombination.These integrated elements can be and hit any sequence of sequence homology of cellular genome.And these integrated elements can be non-coding or code-shaped nucleotide sequence.On the other hand, this carrier can enter in the genome of cell by non-homogeneous recombination and integration.

In the situation of self-replicating, this carrier can further comprise make this carrier can be in bacterial cell and vegetable cell the replication origin of self-replicating.

Host cell

The invention still further relates to the restructuring " host cell " that contains nucleotide sequence of the present invention.The nucleic acid construct or the carrier that contain nucleotide sequence of the present invention can be introduced in the host cell, make nucleotide sequence of the present invention be integrated on the karyomit(e) or make the carrier self-replicating, thereby nucleotide sequence of the present invention is able to stably express in this host cell, causes giving the resistance of the anti-careless glycosides phosphine of this host cell.

Host cell can be the cell of prokaryotic organism such as bacterium, but the more preferably cell of eukaryote such as plant.

Bacterial cell commonly used has the cell of gram positive bacterium such as genus bacillus, or the cell of gram negative bacterium such as intestinal bacteria and pseudomonas.In a preferred embodiment, the colibacillary cell of bacterial host cell.

Expression vector is introduced bacterial host cell can pass through protoplast transformation (such as Chang and Cohen, 1979, molecule General Genetics 168:111-115), utilize competent cell (such as Young and Spizizin, 1961, J.Bacteriol.81:823-829, or Dubnau and Davidoff-Abelson, 1971, J.Mol.Biol.56:209-221), by electroporation (such as Shigekawa and Dower, 1988, Biotech.6:742-751), or pass through conjugation (such as Koehler and Thorne, 1987, J.Bacteriol.169:5771-5278) realize.

Transgenic plant

After allogeneic dna sequence DNA imported vegetable cell, can confirm the integration of heterologous gene in Plant Genome by several different methods known in the art.

The PCR assay method: can be with the specific oligonucleotide primers such as target gene or soil bacillus carrier background be carried out PCR, in order to screen whether there is the gene integrated in the vegetable cell.

The Southern assay method: can confirm Plant Transformation by the Southern trace assay method of genomic dna.Generally speaking, from transformant, extract total DNA, with suitable restriction enzyme digestion, at sepharose separately, and transfer on nitrocellulose or the nylon membrane.Then according to standard technique, detect film or trace with for example radiolabeled target dna fragment, with the integration of foreign gene in Plant Genome of confirming to be imported.

RT-PCR assay method: can confirm heterologous gene transcribing in transgenic plant by the RT-PCR assay method of total RNA.

Western trace assay method: can carry out the Western trace to transgenic plant, wherein using can be in conjunction with the antibody of one or several epi-position on the glyphosate-tolerance albumen.

Embodiment 1: clone, expression and the purifying of pseudomonas putida EPSPS gene aroA

Having used at application number in the present embodiment is disclosed plasmid pKU2002 in 02117991.3 the Chinese patent application, and it is the pUC18 plasmid that comprises pseudomonas putida EPSPS gene aroA wild-type nucleotide sequence (SEQ ID NO:9).

According to the nucleotide sequence of the upper EPSPS gene aroA of plasmid pKU2002, designed and synthesized 5 ' end primer, 1 (5 '-cat Gcc atg gAA GTA ACA ATA CAG CCC GGA G-3 ', SEQ IDNO:1) and 3 ' end primer 2 (5 '-gtt a Ct cga gTG AGA AAT TAA ATT GAT GGT TT-3 ', SEQ ID NO:2) (wherein underscore has indicated NcoI and the XhoI restriction site for the clone in the flank interpolation of aroA nucleotide sequence).Take plasmid pKU2002 as template, pass through pcr amplification aroA nucleotide sequence with primer 1 and primer 2.The PCR condition is: 94 ℃ of denaturations 10 minutes; Then be 30 circulations 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 2 minutes; Being at last 72 ℃ extended 10 minutes again.Gained PCR product is connected into expression vector pET-28a (+) (Novagen), gained plasmid called after pKU2364 after with restriction endonuclease NcoI and XhoI digestion.

Plasmid pKU2364 is transformed into e. coli bl21 (DE3) (Novagen) by Calcium Chloride Method.E. coli bl21 (DE3) inoculation of carrying plasmid pKU2364 is entered the LB liquid nutrient medium that 300mL is supplemented with the 25mg/L kantlex, in 37 ℃ of shaking culture to OD ₆₀₀Reach about 0.8.Adding IPTG is 1mM to final concentration, and continuation was cultivated 3 hours.

Culture in room temperature with 8000rpm centrifugal 10 minutes, is abandoned supernatant liquor, and the bacterial sediment thing is resuspended in the Tris-HCl pH 7.0 that 30mL contains 0.1mM DTT.After the supersound process, use HisTrap HP test kit (Amersham Biosciences) to separate and protein purification according to the specification sheets of manufacturers.Gained protein is pseudomonas putida EPSPS.

Embodiment 2: for the preparation of the polyclonal antibody of pseudomonas putida EPSPS

The pseudomonas putida EPSPS albumen of preparing as described in Example 1 for the rabbit immunization.Behind 4 immunity (use Freund's complete adjuvant for the first time, all the other use Freund's incomplete adjuvant) and 1 booster immunization (use Freund's incomplete adjuvant) (timed interval of each time immunity is 10 days), gather blood and prepare antiserum(antisera).

Embodiment 3: the structure of transgenic constructs

In order in plant, to express EPSPS gene aroA, design and made up transgenic constructs (seeing Fig. 1).On the one hand, according to vegetable codon Preference and nucleic acid secondary structure prediction, the nucleotide sequence of EPSPS gene aroA has been carried out codon optimized, in order to improve the expression level of EPSPS gene aroA in plant and to eliminate possible nucleic acid secondary structure.On the other hand, for the AroA protein priming that will in cell, generate to chloroplast(id), Arabidopis thaliana EPSPS CTP encoding sequence (SEQ ID NO:7) is connected to 5 ' end of the AroA encoding sequence (SEQ ID NO:10) that codon optimizes.

The complete encoding sequence (comprising the pseudomonas putida EPSPS gene aroA nucleotide sequence that Arabidopis thaliana EPSPS CTP nucleotide sequence and codon are optimized) of synthetic is connected into plant expression vector pBI121 (Clontech) by restriction site BamHI and SacI, gained plasmid called after pKU2323.Comprise Arabidopis thaliana EPSPS CTP nucleotide sequence and without the plasmid called after pKU2349 of the pseudomonas putida EPSPS gene aroA nucleotide sequence (SEQ ID NO:9) of optimizing.

Embodiment 4: the structure of transgenic plant

1. prepare the competence agrobacterium tumefaciens

With agrobacterium tumefaciens LBA4404 (Life Technologies) on the LB plate that is supplemented with 50mg/L Streptomycin sulphate and 50mg/L Rifampin in 28 ℃ of activation culture 48 hours.Picking list bacterium colony, inoculation enters the LB liquid nutrient medium that 3mL is supplemented with 50mg/L Streptomycin sulphate and 50mg/L Rifampin, spends the night in 28 ℃ of shaking culture.Culture is inoculated the LB liquid nutrient medium that is supplemented with 50mg/L Streptomycin sulphate and 50mg/L Rifampin into 20mL according to 1: 100 ratio, in 28 ℃ of shaking culture to OD ₆₀₀Reach about 0.6.Culture was placed on ice 10 minutes, then in 4 ℃ with 5000rpm centrifugal 10 minutes, abandon supernatant liquor, the bacterial sediment thing is resuspended in the 2mL 0.2M CaCl of precooling ₂With 2mL 0.1M MgSO ₄Bacterial suspension was placed on ice 30 minutes, then in 4 ℃ with 5000rpm centrifugal 10 minutes, abandon supernatant liquor, the bacterial sediment thing is resuspended in the 0.5mL 0.2M CaCl of precooling ₂With 0.5mL 0.1M MgSO ₄, namely get competent cell.

2. use the recombinant plasmid transformed competence colibacillus Agrobacterium

2ul pKU2323 plasmid DNA (1ug/ul) is added into the 200ul competent cell, and mixing was placed 30 minutes on ice gently.With bacterial suspension cold shock 1 minute in liquid nitrogen, then then immediately heat shock 90 seconds in 42 ℃ of water-baths placed on ice 5 minutes more immediately.Bacterial suspension is inoculated the additional antibiotic LB liquid nutrient medium into 800ul, in 28 ℃ of recovery 4-6 hours.Get an amount of bacterial suspension and be applied on the LB plate that is supplemented with 50mg/L Streptomycin sulphate, 50mg/L Rifampin and 25mg/L kantlex, with plate in 28 ℃ of incubations 2 days.

Picking list bacterium colony from the flat board, inoculation enters the LB liquid nutrient medium that 1mL is supplemented with 50mg/L Streptomycin sulphate, 50mg/L Rifampin and 25mg/L kantlex, spends the night in 28 ℃ of shaking culture.Culture is inoculated the above-mentioned substratum into 50ml, and be cultured to OD ₆₀₀Reach about 0.5 (approximately 3-4 hour).With culture with 5000rpm centrifugal 10 minutes, abandon supernatant liquor, the bacterial sediment thing is resuspended in an amount of MS substratum (Gibco BRL; Also can be referring to Murashige and Skoog, 1962) so that OD ₆₀₀Be 0.2-0.3.

3. infect tobacco with transforming Agrobacterium leaf dish method

Get tobacco (Nicotiana tabacum L.cv.W38) (by professor Lin Zhongping of Peking University present) the bud green young leaflet tablet of color, it is cut into the fragment of 0.8cmx0.8cm, and in above-mentioned bacterial suspension, soaked 15 minutes.With the blade placing that soaked at MS ₁On the substratum (the MS substratum is added 1mg/L 6-benzyladenine and 0.1mg/L naphthylacetic acid), in 25 ℃ of incubations 60 hours in the dark.Then, clean tobacco leaf, and transfer to MS ₂On the substratum (the MS substratum is added 1mg/L 6-benzyladenine, 0.1mg/L naphthylacetic acid, 300mg/L cephamycin and 200mg/L kantlex), in 25 ℃ of incubations 16 hours under illumination.Visible callus forms after 2 weeks, and visible Bud Differentiation grows after 3 weeks.When treating bud length to the 1cm left and right sides, with its cutting-out, and transfer to the MS substratum _1/2Carry out root culture on (the MS substratum is added 0.1mg/L naphthylacetic acid, 300mg/L cephamycin and 200mg/L kantlex), about 1-2 bears adventive root after week.Behind well developed root system, plant is taken out, with the solid medium that the sterilized water flush away adheres to, move in the cultivating soil (the triumphant mattress fertilizer in Beijing limited liability company), cultivate in (25 ℃, 16 hours illumination/8 hour dark) in the greenhouse.

Embodiment 5: the glyphosate resistance assay method of transgenic plant-blade sprays assay method

The transgene tobacco of cultivating in the greenhouse is long during the phase, to spray careless glycosides phosphine weedicide to 6-8 sheet leaf.The used careless glycosides phosphine weedicide of this experiment is Roundup, i.e. 41% careless glycosides phosphine isopropamide AS (being given by About Monsanto Chemicals).With 100 times of Roundup dilutions, evenly be sprayed on (spray value is that per hectare is used about Roundup 3L) on the tobacco leaf.After this observe the tobacco growing situation.

Wild-type adjoining tree (n=5) shows the symptom of being injured after 1-3 days, and stem apex and partial blade begin to wilt; Seriously wilt afterwards and lose green, show withered and yellow, after the week all dead (seeing Fig. 2).The growing state of the blank plant that transforms through empty carrier pBI121 goes out with the wild-type adjoining tree similar.6 strains (being numbered 2323-4,2323-5,2323-15,2323-16,2323-20,2323-23) normal growth is arranged in the 32 strain transfer-gen plants, and all the other are dead gradually.

The transfer-gen plant that the agriculture bacillus mediated conversion of pKU2323 by comprising codon optimization obtains is 2323 series; And the transfer-gen plant that obtains by the agriculture bacillus mediated conversion that comprises without codon optimized pKU2349 is 2349 series.

Embodiment 6: the PCR assay method of transgenic plant, Southern trace assay method and RT-PCR assay method

1. the extraction of tobacco leaf genomic dna

Following Murray and Thompson (1980) revises the CTAB method form and extracts genomic dna from tobacco leaf.Vegetable material was placed 24 hours in the dark, to reduce starch content.The about 100mg blade of clip places mortar.Pour liquid nitrogen into, with the blade grinding powder.The blade powder transfer to the 1.5ml centrifuge tube, is added the CTAB Extraction buffer that 600ul contains 2% mercaptoethanol, mixing.With pipe in 65 ℃ of water-baths incubation 1-1.5 hour.To be cooled to room temperature, add the chloroform that 600ul mixes at 24: 1: primary isoamyl alcohol, mixing is until the blade powder turns white by green.In room temperature with 13,000rpm centrifugal 10 minutes, take out the upper strata water.The chloroform of using again mix in isopyknic 24: 1 water: after the primary isoamyl alcohol extracting once, with 13,000rpm centrifugal 6 minutes.Take out the upper strata water, add the dehydrated alcohol of 2 times of volumes with precipitation DNA.With DNA throw out 70% washing with alcohol, dry.Add an amount of aseptic deionized water that contains the RNA enzyme with the dissolving DNA throw out.

2.PCR assay method

Nucleotide sequence (nucleotide sequence that comprises Arabidopis thaliana EPSPSCTP) according to the EPSPS gene aroA after codon optimized has designed 5 ' end primer 3 (5 '-ATG GCG CAA GTT AGC AGAATC-3 ', SEQ ID NO:3) and 3 ' end primer 4 (5 '-TCA TGA GAA GTT GAA TTGATG-3 ', SEQ ID NO:4).Take the tobacco leaf genomic dna of as above preparation as template, implement PCR with primer 3 and 4.The PCR condition is as follows: 94 ℃ of denaturations 5 minutes; Then be 30 circulations 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes; Being at last 72 ℃ extended 10 minutes again.Detect the PCR product by 0.8% agarose gel electrophoresis.

As shown in Figure 3, transgene tobacco (being numbered 2323-4,2323-5,2323-15,2323-16,2323-20 and 2323-23) that 6 strains tolerate careless glycosides phosphine has all obtained the pcr amplification product that length is about 1.5kb, shows that transgenic constructs ctp::aroA has been incorporated in the tobacco gene group.

3.Southem trace assay method

Get the genomic dna of the positive transgene tobacco of 50ug PCR, with restriction endonuclease EcoRI digestion.DNA after enzyme cut carries out 0.8% agarose gel electrophoresis, and transfers to Hybond by capillary tube technique ^TMOn-N+ the film.

Take the pKU2323 plasmid DNA as template, with primer 3 and primer 5 (5 '-CAAATG TGG AAGAAT TTC ATC-3 ', SEQ ID NO:5) implements PCR, obtained the amplified production of 615bp, specifically comprise front 130 the amino acid whose encoding sequences of whole CTP and AroA.Use efficient DNA digoxigenin labeled and detection kit (DIG-High Prime DNA Labeling and Detection Starter Kit I) (Roche) according to the specification sheets of manufacturers to digoxin on the PCR Product Labeling, and carry out hybridization according to ordinary method in 42 ℃.

As shown in Figure 4, in the transgene tobacco of the careless glycosides phosphine of tolerance, goal gene only has a copy in the tobacco gene group, and does not hybridize to goal gene in WT contrast tobacco plant.

4.RT-PCR assay method

Choose the fresh blade of 100mg, use RNAprep pure plant total RNA extraction reagent box (Tiangen) to extract RNA according to the specification sheets of manufacturers.After using M-MLV reversed transcriptive enzyme (Promega) to prepare cDNA according to the specification sheets of manufacturers, implement PCR with primer 3 and 4.The PCR condition is as follows: 94 ℃ of denaturations 5 minutes; Then be 30 circulations 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes; Being at last 72 ℃ extended 10 minutes again.Detect the PCR product by 0.8% agarose gel electrophoresis.

As shown in Figure 5, transgene tobacco (being numbered 2323-4,2323-5,2323-15,2323-16,2323-20 and 2323-23) that 6 strains tolerate careless glycosides phosphine has all obtained the RT-PCR amplified production that length is about 1.5kb, shows that transgenosis carried out effectively transcribing in transgene tobacco.

As shown in Figure 6, the RT-PCR assay method shows that also the transgenosis of codon optimization has improved the stability of this genetically modified mRNA in transgene tobacco significantly.

Embodiment 7: the Western trace assay method of transgenic plant

Get the fresh tobacco leaf of 100mg, add liquid nitrogen and fully grind, powder transfer is entered the 2ml centrifuge tube.After liquid nitrogen volatilizees fully, add 200ul proteins extraction damping fluid (0.25M Tris-HCl pH 6.8,8% mercaptoethanols, 20% glycerine, 8%SDS), behind the vibration mixing in 100 ℃ of incubations 8 minutes.With mixture with 12000rpm centrifugal 15 minutes, take out supernatant liquor, be the leaf protein crude product.

Prepare the SDS-PAGE gel according to routine techniques, it concentrates gum concentration at the middle and upper levels is 5%, and lower floor's resolving gel concentration is 16%.After protein carried out SDS-PAGE, the transfer printing mark was to nitrocellulose filter.With the anti-EPSPS polyclonal antiserum hybridization of the rabbit of film and dilution in 1: 2000.Then, the coupling with Antibody-antigen complex and dilution in 1: 3000 has anti-rabbit igg of goat (Promega) reaction of horseradish peroxidase to form ternary complex.Add DAB solution (PIERCE) with colour developing.

As shown in Figure 7, the molecular weight of EPSPS albumen is about 47kDa.In the transgene tobacco of the careless glycosides phosphine of tolerance, detected EPSPS albumen.In the transgene tobacco that does not tolerate careless glycosides phosphine, although the existence that has detected the aroA gene by PCR can't detect EPSPS albumen (result does not show) by Western.

Embodiment 8: transgenic plant T1 is for the glyphosate resistance assay method of seedling-seed germination assay method

In order to detect inheritability and the stability of EPSPS gene aroA in the process of going down to posterity, T0 has been carried out the seed germination assay method for the seed of plant, detect T1 for the tolerance of seedling to careless glycosides phosphine.

As shown in Figure 8, preliminary experiment has determined that making the minimum careless glycosides phosphine concentration of the complete albefaction death of wild-type tobacco is 0.5mM.With seed-coat sterilization (70% ethanol 30 second of transgene tobacco T0 for plant, 20% chlorine bleach liquor 30 minutes, the sterilized water washing for several times), be inoculated on the MS substratum that contains different concns (0,0.1mM and 1mM) careless glycosides phosphine (Sigma), cultivate in the dark 3 days to sprout in 25 ℃, transfer illumination condition to again and cultivate, 3-4 observes the growth of seedling situation after week.

As shown in Figure 9, when careless glycosides phosphine concentration was 1mM, most 2323 was well rudiment and the growth of seed of transgene tobacco; And empty carrier contrasts the seed germination of tobacco but can not survive, and slowly albefaction is dead.When careless glycosides phosphine concentration was 0mM, 2323 was all well rudiment and growths of seed of transgene tobacco and empty carrier contrast tobacco.As shown in figure 10, when careless glycosides phosphine concentration was 10mM, T1 still can sprout growth for seedling.

In addition, on the flat board that contains careless glycosides phosphine, the transfer-gen plant Seed germination that tolerates careless glycosides phosphine shows two kinds of situations: except most seeds can be good at sprouting and growth; Also have the minority seed to show the situation of similar empty map plant seed, can germinate but can not grow but albefaction is dead at leisure.Table 1 is that 6 strains are tolerated the seed germination of transgene tobacco of careless glycosides phosphine and the mathematical statistics result of growth.Wherein, the ratio that the T1 seedling of same plant shows tolerance and susceptibility to careless glycosides phosphine met mendel's law (Mendelian Law) near 3: 1.

Table 1: the T1 of transfer-gen plant is for tolerance and the susceptibility of seedling to careless glycosides phosphine

T1 has been carried out the PCR detection as mentioned above for the plant that seedling grows up to.As shown in figure 10, PCR result shows the existence that can detect the purpose fragment in the seedling that careless glycosides phosphine is tolerated; Then do not detect the existence of purpose fragment in the seedling to careless glycosides phosphine sensitivity.

Sequence table

＜110〉Peking University

＜120〉transgenic constructs and transgenic plant

<160>10

<170>PatentIn version 3.1

<210>1

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>1

CAT GCC ATG GAA GTA ACA ATA CAG CCC GGA G

<210>2

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

GTT ACT CGA GTG AGA AAT TAA ATT GAT GGT TT

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

ATG GCG CAA GTT AGC AGA ATC

<210>4

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>4

TCA TGA GAA GTT GAA TTG ATG

<210>5

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>5

CAA ATG TGG AAG AAT TTC ATC

<210>6

<211>75

<212>PRT

＜213〉Arabidopis thaliana

<220>

＜223〉aminoacid sequence of Arabidopis thaliana EPSPS chloroplast transit peptides

<400>6

MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG LKKSGMTLIG

SELRPLKVMS SVSTA

<210>7

<211>225

<212>DNA

＜213〉Arabidopis thaliana

<220>

＜223〉nucleotide sequence of Arabidopis thaliana EPSPS chloroplast transit peptides

<400>7

ATGGCGCAAG TTAGCAGAAT CTGCAATGGT GTGCAGAACC CATCTCTTAT CTCCAATCTC

TCGAAATCCA GTCAACGCAA ATCTCCCTTA TCGGTTTCTC TGAAGACGCA GCAGCATCCA

CGAGCTTATC CGATTTCGTC GTCGTGGGGA TTGAAGAAGA GTGGGATGAC GTTAATTGGC

TCTGAGCTTC GTCCTCTTAA GGTCATGTCT TCTGTTTCCA CGGCG

<210>8

<211>431

<212>PRT

＜213〉pseudomonas putida

<220>

＜223〉aminoacid sequence of pseudomonas putida EPSPS gene aroA coding

<400>8

EKVTIQPGDL TGIIQSPASK SSMQRACAAA LVAKGISEII NPGHSNDDKA ARDIVSRLGA

RLEDQPDGSL QITSEGVKPV APFIDCGESG LSIRMFTPIV ALSKEEVTIK GSGSLVTRPM

DFFDEILPHL GVKVKSNQGK LPLVIQGPLK PADVTVDGSL SSQFLTGLLL AYAAADASDV

AIKVTNLKSR PYIDLTLDVM KRFGLKTPEN RNYEEFYFKA GNVYDETKMQ RYTVEGDWSG

GAFLLVAGAI AGPITVRGLD IASTQADKAI VQALMSANAG IAIDAKEIKL HPADLNAFEF

DATDCPDLFP PLVALASYCK GETKIKGVSR LAHKESDRGL TLQDEFGKMG VEIHLEGDLM

RVIGGKGVKG AEVSSRHDHR IAMACAVAAL KAVGETTIEH AEAVNKSYPD FYSDLKQLGG

VVSLNHQFNF S

<210>9

<211>1293

<212>DNA

＜213〉pseudomonas putida

<220>

＜223〉the wild-type nucleotide sequence of pseudomonas putida EPSPS gene aroA

<400>9

ATGCAAGTAA CAATACAGCC CGGAGATCTG ACTGGAATTA TCCAGTCACC CGCTTCAAAA

AGTTCGATGC AGCGAGCTTG TGCTGCTGCA CTGGTTGCAA AAGGAATAAG TGAGATCATT

AATCCCGGTC ATAGCAATGA TGATAAAGCT GCCAGGGATA TTGTAAGCCG GCTTGGTGCC

AGGCTTGAAG ATCAGCCTGA TGGTTCTTTG CAGATAACAA GTGAAGGCGT AAAACCTGTC

GCTCCTTTTA TTGACTGCGG TGAATCTGGT TTAAGTATCC GGATGTTTAC TCCGATTGTT

GCGTTGAGTA AAGAAGAGGT GACGATCAAA GGATCTGGAA GCCTTGTTAC AAGACCAATG

GATTTCTTTG ATGAAATTCT TCCGCATCTC GGTGTAAAAG TTAAATCTAA CCAGGGTAAA

TTGCCTCTCG TTATACAGGG GCCATTGAAA CCAGCAGACG TTACGGTTGA TGGGTCCTTA

AGCTCTCAGT TCCTTACAGG TTTGTTGCTT GCATATGCGG CCGCAGATGC AAGCGATGTT

GCGATAAAAG TAACGAATCT CAAAAGCCGT CCGTATATCG ATCTTACACT GGATGTGATG

AAGCGGTTTG GTTTGAAGAC TCCCGAGAAT CGAAACTATG AAGAGTTTTA TTTCAAAGCC

GGGAATGTAT ATGATGAAAC GAAAATGCAA CGATACACCG TAGAAGGCGA CTGGAGCGGT

GGTGCTTTTT TACTGGTAGC GGGGGCTATT GCCGGGCCGA TCACGGTAAG AGGTTTGGAT

ATAGCTTCGA CGCAGGCTGA TAAAGCGATC GTTCAGGCTT TGATGAGTGC GAACGCAGGT

ATTGCGATTG ATGCAAAAGA GATCAAACTT CATCCTGCTG ATCTCAATGC ATTTGAATTT

GATGCTACTG ATTGCCCGGA TCTTTTTCCG CCATTGGTTG CTTTGGCGTC TTATTGCAAA

GGAGAAACAA AGATCAAAGG CGTAAGCAGG CTGGCGCATA AAGAAAGTGA CAGAGGATTG

ACGCTGCAGG ACGAGTTCGG GAAAATGGGT GTTGAAATCC ACCTTGAGGG AGATCTGATG

CGCGTGATCG GAGGGAAAGG CGTAAAAGGA GCTGAAGTTA GTTCAAGGCA CGATCATCGC

ATTGCGATGG CTTGCGCGGT GGCTGCTTTA AAAGCTGTGG GTGAAACAAC CATCGAACAT

GCAGAAGCGG TGAATAAATC CTACCCGGAT TTTTACAGCG ATCTTAAACA ACTTGGCGGT

GTTGTATCTT TAAACCATCA ATTTAATTTC TCA

<210>10

<211>1293

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide sequence of the codon of pseudomonas putida EPSPS gene aroA optimization

<400>10

GAGAAAGTGA CAATCCAACC AGGAGATCTG ACCGGAATCA TCCAATCACC AGCTTCAAAA

AGCTCCATGC AaaGAGCTTG TGCTGCTGCt tTGGTTGCtA AgGGAATttc TgaaATtATT

AAcCCaGGTC ATAGCAAcGA TGATAAgGCT GCtAGaGATA TTGTtAGCaG aCTTGGTGCt

AGaCTTGAAG ATCAaCCTGA TGGTTCTTTG CAaATtACtt cTGAAGGaGT tAAgCCTGTt

GCTCCTTTcA TTGAtTGCGG TGAATCTGGT TTgtcTATta GaATGTTcAC TCCaATTGTT

GCtTTGtcTA AgGAAgaaGT GACtATtAAg GGATCTGGAA GCCTTGTTAC tAGACCAATG

GATTTCTTcG ATGAAATTCT TCCaCATtTg GGTGTtAAgG TTAAgTCTAA CCAaGGTAAg

TTGCCTtTgG TTATtCAaGG aCCATTGAAg CCAGCtGAtG TTACtGTTGA TGGaTCtTTg

TCTTCTCAaT TCCTTACtGG TTTGTTGCTT GCtTAcGCtG CtGCtGATGC tAGCGATGTT

GCtATtAAgG TtACtAActT gAAgAGCCGT CCaTAcATtG ATCTTACttT GGATGTGATG

AAGaGaTTcG GTTTGAAGAC TCCaGAGAAc aGAAACTAcG AAgaaTTcTA cTTCAAgGCt

GGaAAcGTtT AcGATGAAAC tAAgATGCAA aGATACACCG TtGAAGGaGA tTGGAGCGGT

GGTGCTTTcT TgtTGGTtGC tGGaGCTATT GCtGGaCCaA TtACtGTtAG AGGTTTGGAT

ATtGCTTCGA CtCAaGCTGA TAAgGCtATt GTTCAaGCTT TGATGtcTGC tAACGCtGGT

ATTGCtATTG ATGCtAAgga aATtAAgCTT CATCCTGCTG ATtTgAAcGC tTTcGAATTc

GATGCTACTG ATTGCCCaGA TCTTTTcCCa CCATTGGTTG CTTTGGCtTC TTAcTGCAAg

GGAGAAACtA AGATtAAgGG aGTtAGCAGa tTGGCtCATA AgGAAtcTGA tAGAGGATTG

ACttTGCAaG AtgaaTTCGG aAAgATGGGT GTTGAAATtC ACCTTgaaGG AGATtTGATG

aGaGTGATtG GAGGaAAgGG aGTtAAgGGt GCTGAAGTTt cTTCAAGaCA CGATCATaGa

ATTGCtATGG CTTGCGCtGT GGCTGCTTTg AAgGCTGTGG GTGAAACtAC CATtGAACAT

GCtGAAGCtG TGAAcAAgTC tTACCCaGAT TTcTACAGCG ATCTTAAgCA ACTTGGaGGT

GTTGTtTCTT TgAACCATCA ATTcAAcTTC TCA

Claims (14)

1. the polynucleotide of a separation, its direction with from 5 ' to 3 ' is comprised of the nucleotide sequence of coding CTP and the nucleotide sequence of the careless glycosides phosphine tolerance type EPSPS of coding, the nucleotide sequence of wherein said coding CTP is the nucleotide sequence of coding Arabidopis thaliana EPSPS CTP, and the nucleotide sequence of the careless glycosides phosphine of described coding tolerance type EPSPS is the nucleotide sequence of coding pseudomonas putida EPSPS, and the aminoacid sequence of wherein said Arabidopis thaliana EPSPS CTP is SEQ ID NO:6, and the aminoacid sequence of described pseudomonas putida EPSPS is SEQ ID NO:8.

2. polynucleotide claimed in claim 1, the nucleotide sequence of wherein said coding Arabidopis thaliana EPSPS CTP is SEQ ID NO:7.

3. claim 1 or 2 described polynucleotide, the nucleotide sequence of wherein said coding pseudomonas putida EPSPS is SEQ ID NO:9.

4. claim 1 or 2 described polynucleotide, the nucleotide sequence of wherein said coding pseudomonas putida EPSPS are the nucleotide sequences of the coding pseudomonas putida EPSPS that optimizes of codon.

5. polynucleotide claimed in claim 4, the nucleotide sequence of the coding pseudomonas putida EPSPS of wherein said codon optimization is SEQ ID NO:10.

6. nucleic acid construct, it comprises the polynucleotide of each described separation of claim 1-5.

7. recombinant vectors, it comprises the polynucleotide of each described separation of claim 1-5, or comprises nucleic acid construct claimed in claim 6.

8. recombinant host cell, it comprises the polynucleotide of each described separation of claim 1-5, or comprises nucleic acid construct claimed in claim 6, or comprises recombinant vectors claimed in claim 7, and wherein said recombinant host cell is bacterial cell.

9. the recombinant host cell of claim 8, wherein said recombinant host cell is Agrobacterium.

10. the recombinant host cell of claim 9, wherein said recombinant host cell is agrobacterium tumefaciens.

11. the recombinant host cell of claim 8, wherein said recombinant host cell are intestinal bacteria.

12. prepare the method for transgenic plant cells and transgenic plant, it comprises the step that each described recombinant host cell of the polynucleotide of each described separation of claim 1-5, nucleic acid construct claimed in claim 6, recombinant vectors claimed in claim 7 or claim 8-10 is imported vegetable cell.

13. give vegetable cell or the plant method with careless glycosides phosphine tolerance, it comprises the step that each described recombinant host cell of the polynucleotide of each described separation of claim 1-5, nucleic acid construct claimed in claim 6, recombinant vectors claimed in claim 7 or claim 8-10 is imported vegetable cell.

14. fusion rotein, it is comprised of CTP and careless glycosides phosphine tolerance type EPSPS with the direction of holding the C end from N, wherein said CTP is Arabidopis thaliana EPSPS CTP, and described careless glycosides phosphine tolerance type EPSPS is pseudomonas putida EPSPS, and the aminoacid sequence of wherein said Arabidopis thaliana EPSPS CTP is SEQ ID NO:6, and the aminoacid sequence of described pseudomonas putida EPSPS is SEQ ID NO:8.

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